Activated protein C modulates inflammation, apoptosis and tissue factor procoagulant activity by regulating endoplasmic reticulum calcium depletion in blood monocytes

Background: The endoplasmic reticulum (ER) is responsible for the synthesis and folding of secretory, transmembrane and ER-resident proteins. Conditions that impair protein folding or overwhelm its protein folding capacity disrupt ER homeostasis, thereby causing ER stress. ER stress-induced apoptosis and inflammation are involved in the pathogenesis of inflammatory diseases. Activated protein C (APC) inhibits inflammation and apoptosis in monocytes, and this may partly explain the protective effects of APC treatment in severe sepsis. However, the precise molecular pathways by which APC modulates these effects remain unknown. Objectives: To investigate whether APC modulates the ER stress response in human monocytes. Methods: We treated monocytes with ER stress-inducing agents in the presence or absence of APC to determine the effect on this response. Protein and mRNA levels were determined by immunoblotting and real-time PCR, respectively. Enzyme assays and flow cytometry were used to determine the role of APC in this model. Results: In thapsigargin (Tg)-treated cells, APC dampened unfolded protein response activation, as indicated by reduced levels of the 78-kDa glucose-regulated protein (GRP78), in an endothelial protein C receptor-independent and protease-activated receptor-1-independent manner. Consistent with this, APC decreased phosphorylated eukaryotic translational initiation factor 2 alpha and C/EBP homologous protein levels induced by Tg. APC inhibited Tg-induced ER Ca2+ flux and reactive oxygen species generation. Functionally, APC diminished Tg-induced caspase-3 activity and degradation of the nuclear factor kappaB inhibitor I kappa B alpha. Furthermore, APC dampened the induction of tissue factor procoagulant activity facilitated by Tg. Conclusions: These studies suggest that APC modulates the adverse effects of ER Ca2+ depletion in human monocytes.